3-Aminomethylene-2,4-pentanedione bis(thiosemicarbazone) derivatives

ABSTRACT

A radioactive diagnostic agent which comprises a physiologically active substance and a radioactive metallic element combined with a compound of the formula: ##STR1## wherein R 1  and R 2  are each hydrogen, C 1  -C 3  alkyl or phenyl. The agent is characteristic in having a high stability even after being administered into a human body and showing the substantially the same behavior as the physiologically active substance itself in a human body.

The present invention relates to 3-aminomethylene-2,4-pentanedionebis(thiosemicarbazone) derivatives, and their production and use. Moreparticularly, it relates to 3-aminomethylene-2,4-pentanedionebis(thiosemicarbazone) derivatives (hereinafter referred to as "BTS") ofthe formula: ##STR2## wherein R¹ and R² are each a hydrogen atom, a C₁-C₃ alkyl group or a phenyl group, their production process and theiruse as a carrier for a radioactive metallic element as well aphysiologically active substance.

For the purpose of a non-invading nuclear medical diagnosis such asrecording, dynamic study and quantitative measurement of the bloodcirculation system, detection of physiological abnormality orlocalization of abnormality by imaging, there have been widely usedphysiologically active substances labeled with iodine-131 (¹³¹ I) suchas ¹³¹ I-labeled serum albumin and ¹³¹ I-labeled fibrinogen. However,¹³¹ I has a long half life of about 8 days and emits beta-rays so thatthe patient administered therewith is exposed to a large quantity ofradiation.

In order to overcome the said drawback in the ¹³¹ I-labeledphysiologically active substances, attempts have been made to provideradioactive diagnostic agents comprising physiologically activesubstances and radioactive metallic elements having more favorablephysical properties than iodine-131 combined thereto. Among suchattempts, there is known a labeling method wherein a physiologicallyactive substance is treated directly with a radioactive metal salt tomake a chelate compound, which may be used as a radioactive diagnosticagent. For instance, human serum albumin is treated with an aqueoussolution containing technetium-99m (^(99m) Tc) in the form ofpertechnetate in the presence of a reducing agent to give ^(99m)Tc-labeled human serum albumin. Further, for instance, bleomycin istreated with an aqueous solution containing indium-111 (¹¹¹ In) in theform of indium chloride to give ¹¹¹ In-labeled bleomycin. However, thechelate forming property of those physiologically active substances isnot sufficient, and the once formed chelating bond is readily broken. Infact, ^(99m) Tc-labeled serum albumin and ¹¹¹ In-labeled bleomycin arelow in the stability after administration into living bodies so that thebehavior of the radioactivity in such bodies does not coincide with thatof serum albumin or bleomycin as the physiologically active substance.This is a fatal defect for the nuclear medical diagnosis based on theexact trace of the behavior of the radioactivity which should coincidewith the behavior of the physiologically active substance.

As a result of an extensive study, it has now been found that the BTS(I) has a strong chelate-forming property and can be bonded to an aminogroup and/or a carboxyl group in physiologically active substances undera mild condition. It has also been found that a chemical productcomprising a physiologically active substance and a radioactive metallicelement bonded thereto with intervention of the BTS (I) is sufficientlystable in living bodies, and the behavior of the radioactivity in livingbodies is quite coincident with that of the physiologically activesubstance itself.

According to the present invention, there is provided the BTS (I), whichis useful as a chemical carrier for a physiologically active substanceand a radioactive metallic element. There is also provided thephysiologically active substance-combined BTS (I) comprising the BTS (I)and a physiologically active substance chemically bonded thereto with orwithout intervention of any linking aid, which is useful as anon-radioactive carrier to be used in diagnosis in nuclear medicine.There is further provided the radioactive metallic element-labeled,physiologically active substance-combined BTS (I) comprising thephysiologically active substance-combined BTS (I) and a radioactivemetallic element chelated thereto, which is useful as a radioactivediagnostic agent.

The BTS (I) is novel and can be produced by condensing3-aminomethylene-2,4-pentadione (A. Kreutzberger et al.: J.Org.Chem.,26, 1121 (1961); K. R. Huffman et al.: J.Org.Chem., 27 551 (1962)) with4-R-thiosemicarbazide (wherein R is the same as R¹ and R²). Thecondensation may be carried out in a single step or in two steps. Whenthe BTS (I) wherein R¹ and R² are same is to be produced, thecondensation is usually carried out in a single step by reacting3-aminomethylene-2,4-pentadione with 4-R-thiosemicarbazide in a molarproportion of 1:2 or more. When the BTS (I) wherein R¹ and R² aredifferent is to be produced, the condensation is ordinarily carried outin two steps by reactig 3-aminomethylene-2,4-pentadione with 4-R¹-thiosemicarbazide in a nearly equimolar proportion and then reactingthe resultant monothiosemicarbazone with 4-R² -thiosemicarbazide in anearly equimolar proportion. In general, the condensation is effected inthe presence of an acidic catalyst such as hydrochloric acid,hydrobromic acid or sulfuric acid, preferably in an inert solvent suchas methanol or ethanol.

The BTS (I) thus produced has two thiosemicarbazone groups which cancatch a radioactive metallic element to form a chelate and an aminogroup which can be bonded to a carboxyl group or an amino group in aphysiologically active substance with or without intervention of anylinking aid under a mild condition to fix such physiologically activesubstance firmly. Therefore, it is useful as a carrier for theradioactive metallic element and the physiologically active substance.

For manufacture of the radioactive diagnostic agent of the presentinvention, the BTS (I) is usually first combined with a physiologicallyactive substance, and then the resultant combined product is labeledwith a radioactive metallic element.

The term "physiologically active substance" is intended to mean anysubstance which can show a specific accumulability at a certain organ ortissue or a certain diseased locus or exhibits a specific behaviorcorresponding to a certain physiological state. Tracing of its behaviorin a living body can provide informations useful for diagnosis. Suchphysiologically active substance as having a carboxyl group or an aminogroup is usable advantageously in this invention. Even when a carboxylgroup or an amino group is not present, it may be used by introducingpreviously a carboxyl group or an amino group therein. Specific examplesof the physiologically active substance are blood proteins (e.g. humanserum albumin, fibrinogen), enzymes (e.g. urokinase, streptokinase),hormones (e.g. thyroid stimulating hormone, parathyroid hormone), immuneantibodies (e.g. IgG), antibiotics (e.g. bleomycin, kanamycin),saccharides, fatty acids, amino acids, etc.

The combination of the BTS (I) with a physiologically active substancemay be carried out according to any procedure as conventionally adoptedfor linking an amino group with a carboxyl group or an amino group.Examples of such procedure include the carbodiimide process, theglutaraldehyde process, etc. According to the carbodiimide process, theBTS (I) having an amino group and a physiologically active substancehaving a carboxyl group are condensed in the presence of a carbodiimidesuch as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form a carbonamidelinkage between the amino group and the carboxyl group. According to theglutaraldehyde process, the BTS (I) having an amino group and aphysiologically active substance having an amino group are reacted inthe presence of glutaraldehyde as a linking aid, and the resultantSchiff base is reduced with a reducing agent such as sodium borohydride.In the resulting products, two amino groups are combined withintervention of a pentamethylene linkage. These bonding procedures arequite advantageous in accomplishment of the bonding under a mildcondition so that any inactivation, denaturation or decomposition of thephysiologically active substance does not materially take place.

When desired, the thus prepared physiologically activesubstance-combined (hereinafter referred to as "PAS-combined") BTS (I)may be purified by a per se conventional procedure such as dialysis, gelfiltration or column chromatography so as to eliminate impurities suchas unreacted reagents therefrom. As the result, the combined product isusually obtained in the form of an aqueous solution, and this aqueoussolution may be as such used for labeling with a radioactive metallicelement. Alternatively, the aqueous solution may be subjected tolyophilization, evaporation under reduced pressure at low temperaturesor the like to obtain a dried product, which also can be used as such orin the form of solution for labeling. Depending on the use, the saidaqueous solution or the said dried product may be incorporated with anyadditive such as a pH controlling agent (e.g. an acid, a base, abuffer), a stabilizer (e.g. ascorbic acid), an isotonizing agent (e.g.sodium chloride) or a preserving agent (e.g. benzyl alcohol). Inaddition, the said aqueous solution or the said dried product maycontain any reducing or oxidizing agent, which will act on a radioactivemetallic element to be labeled so as to give a stable chelate product,as hereinafter explained. Still, the PAS-combined BTS (I) is per sequite stable and can be readily labeled with a radioactive metallicelement by a simple procedure as hereinafter explained, and therefore itmay be stored and supplied on the demand so that its production from theBTS (I) and the physilogically active substance can be saved from thepractitioner such as a medical doctor.

For the labeling of the PAS-combined BTS (I) as the non-radioactivecarrier with a radioactive metallic element, the PAS-combined BTS (I)may be treated with the radioactive metallic element in an appropriateform.

The term "radioactive metallic element" is intended to mean any metallicelement having radioactivity, which has physical characteristicssuitable for nuclear medical diagnosis. Specific examples of theradioactive metallic element are gallium-67 (⁶⁷ Ga), gallium-68 (⁶⁸ Ga),thallium-201 (²⁰¹ Tl), ¹¹¹ In, ^(99m) Tc, etc. They are normallyemployed in their salt forms, particularly in their water-soluble saltforms.

Depending upon the kind or state of the radioactive metallic element,there may be adopted two different labeling manners. When theradioactive metallic element is in a valency state which is not requiredto be reduced or oxidized for formation of a stable chelate compound,the PAS-combined BTS (I) is contacted with the radioactive metallicelement in an aqueous medium to obtain the radioactive metallicelement-labeled, PAS-combined BTS (I). This labeling manner may beapplied to ⁶⁷ Ga, ¹¹¹ In, etc. When the radioactive metallic element isin a valency state which is required to be reduced or oxidized forformation of a stable chelate compound, the PAS-combined BTS (I) iscontacted with the radioactive metallic element in an aqueous medium inthe presence of a reducing agent or an oxidizing agent to obtain theradioactive metallic element-labeled, PAS-combined BTS (I). Thislabeling manner may be applied to ^(99m) Tc, etc.

As the reducing agent, there may be usually employed a stannous salt,i.e. a salt of divalent tin ion (Sn⁺⁺). Specific examples are stannoushalides (e.g. stannous chloride, stannous fluoride), stannous sulfate,stannous nitrate, stannous acetate, stannous citrate, etc. Sn⁺⁺ion-bearing resins such as ion-exchange resins charged with Sn⁺⁺ ion arealso usable.

When, for instance, the radioactive metallic element is ^(99m) Tc, thePAS-combined BTS (I) may be treated with ^(99m) Tc in the form ofpertechnetate in an aqueous medium in the presence of a reducing agentsuch as a stannous salt. As to the order of the introduction of theabove reagents into the reaction system, any particular limitation doesnot exist. Usually, however, the mixing of the stannous salt with thepertechnetate in an aqueous medium in the first place should be avoided.The stannous salt may be used in such an amount as can reducesufficiently the pertechnetate.

The radioactive diagnostic agent should have sufficient radioactivityand radioactivity concentration which can assure reliable diagnosis. Forinstance, in case of the radioactive metallic element being ^(99m) Tc,it may be included usually in an amount of 0.1 to 50 mCi in about 0.5 to5.0 ml at the time of administration. The amount of the PAS-combined BTS(I) may be such as sufficient to form a stable chelate compound with theradioactive metallic element.

The thus produced radioactive metallic element-labeled, PAS-combined BTS(I) as the radioactive diagnostic agent is sufficiently stable, andtherefore it may be stored as such as supplied on the demand. Whendesired, the radioactive diagnostic agent may contain any additive suchas a pH controlling agent (e.g. an acid, a base, a buffer), a stabilizer(e.g. ascorbic acid) or an isotonizing agent (e.g. sodium chloride).

The radioactive metallic element-labeled, PAS-combined BTS (I) of thisinvention is useful for nuclear medical diagnosis. For instance, ^(99m)Tc-labeled, human serum albumin-combined BTS (I) can be used forrecording, dynamic study and quantitative measurement of the bloodcirculation system by administering intravenously to a human body.Further, for instance, ^(99m) Tc-labeled, fibrinogen-combined BTS (I) or^(99m) Tc-labeled, urokinase-combined BTS (I) may be used for detectionand recording of thrombosis as well as localization of thrombosis, sincethey accumulate at the locus of thrombosis. Further, for instance,^(99m) Tc-labeled, streptokinase-combined BTS (I) is useful fordetermination of the locus of myocardial infarction. Moreover, forinstance, ^(99m) Tc-labeled, thyroid stimulating hormone-combined BTS(I) is useful for detection and recording of a cancer at the thyroidgland.

The radioactive diagnostic agent of this invention may be administeredto patients in an amount sufficient to produce a radioactivity necessaryfor examination of the organ or tissue by an appropriate route, usuallythrough an intravenous route. For instance, the intravenousadministration of a ^(99m) Tc-labeled radioactive diagnostic agent ofabout 1 to 3 ml in volume having a radioactivity of about 1 to 20 mCi toa patient is quite suitable for the diagnostic purpose.

The advantages of the PAS-combined BTS (I) as a non-radioactive carriermay be summarized as follows: (a) it is stable over a long period oftime after manufacture; (b) since it can be produced under a mildcondition, any unfavorable side reaction such as inactivation,denaturation or decomposition is not materially caused to thephysiologically active substance; (c) any physiologically activesubstance having a carboxyl group or an amino group is usable as thestarting material; (d) even when a carboxyl group or an amino group isnot present, the introduction of such group into a physiologicallyactive substance makes it usable as the starting material; (e) by such asimple procedure as contacting with a radioactive metallic element in anaqueous medium, it can afford a radioactive metallic element-labeled,PAS-combined BTS (I). Likewise, the advantages of the radioactivemetallic element-labeled, PAS-combined BTS (I) as a radioactivediagnostic agent may be summarized as follows: (a) it is stable over along period of time after manufacture; (b) the labeling efficiency withthe radioactive metallic element is extremely high (nearly 100%); (c)since the labeling operation is quite simple, any unfavorable sidereaction such as inactivation, denaturation or decomposition is notcaused to the physiologically active substance bonded to the BTS (I);(d) among various radioactive metallic elements, the most suitable onefor the diagnostic purpose may be chosen and used so that theinformations for diagnosis is enhanced not only in quantity but also inquality with reduction of the exposure dose.

Practical and presently preferred embodiments of the invention areillustratively shown in the following Examples wherein % is by weight,unless otherwise defined.

EXAMPLE 1 Production of 3-aminomethylene-2,4-pentanedionebis(N-methylthiosemicarbazone) (hereinafter referred to as "BMTS")

A solution of syn-triazine (1 g; 12.3 mmol) in acetylacetone (9.3 g;92.5 mmol) was heated at 155° C. for about 30 minutes. The reactionmixture was cooled to room temperature, whereby pale orange crystalsprecipitated. The crystals were collected by filtration. The filtratewas concentrated and allowed to stand at room temperature, wherebyadditional crystals precipitated. The additional crystals were collectedby filtration, combined with the previously collected crystals andrecrystallized from methanol to give 3-aminomethylene-2,4-pentanedione(2.96 g).

M.P., 146° C.

3-Aminomethylene-2,4-pentanedione (530 mg; 4.2 mmol) as above obtainedand 4-methylthiosemicarbazide (888 mg; 8.5 mmol) were dissolved inmethanol (300 ml), 12N hydrochloric acid (0.3 ml) was added thereto, andthe resultant mixture was heated at 40° C. while stirring for 7 to 8hours. The precipitated crystals were collected by filtration andrecrystallized from methanol to give BMTS (493 mg). M.P., 239° C.(decomp.).

Anal. Calcd. for C₁₀ H₁₉ N₇ S₂ : C, 39.85%; H, 6.35%; N, 32.53%; S,21.27%. Found: C, 40.16%; H, 5.80%; N, 31.61%; S, 21.50%.

EXAMPLE 2 Production of 3-aminomethylene-2,4-pentanedionebis(N-ethylthiosemicarbazone) (hereinafter referred to as "BETS")

In the same manner as in Example 1 but using 4-ethylthiosemicarbazide inplace of 4-methylthiosemicarbazide, there was produced BETS.

EXAMPLE 3 Preparation of human serum albumin-combined BMTS as anon-radioactive carrier

On an ice bath, human serum albumin (lyophilized; 75 mg) was dissolvedin water (5 ml) to give the solution (A). Separately, BMTS was dissolvedin dimethylformamide to make a concentration of 4 mg/ml. To 0.5 ml ofthe resultant solution, the solution (A) was added, and the resultantmixture was adjusted with 0.1N hydrochloric acid to a pH of 90 mg) wasdissolved in 0.01M phosphate buffer-0.15M sodium chloride solution (pH,7.4; 10 ml) to make the solution (B). At a temperature of 0° to 4° C.,the solution (A) (1.0 ml) was added to the solution (B), and theresultant mixture was stirred at the same temperature as above for about1 hour. After addition of sodium borohydride (1 mg), stirring wascontinued at a temperature of 0° to 4° C. for about 1 hour, wherebyreduction proceeded. The resultant mixture was admitted in a dialyzingtube and subjected to dialysis in a conventional manner for 24 hours.The resulting solution was passed through a filter having a pore size of0.22 μm, and 1.0 ml of the filtrate was filled in a vial, followed bylyophilization to obtain a non-radioactive carrier. The above operationswere effected under sterile conditions.

When dissolved in water, the non-radioactive carrier gave a slightlypale yellow, transparent solution.

EXAMPLE 5 Preparation of urokinase-combined BMTS as a non-radioactivecarrier

On an ice bath, purified urokinase (lyophilized; 60 mg) was dissolved inwater (5 ml) to give the solution (A). Separately, BMTS was dissolved indimethylformamide to make a concentration of 4 mg/ml. To 0.5 ml of theresultant solution, the solution (A) was added, and the pH was adjustedwith 0.1N hydrochloric acid to about 4.6 to make the solution (B). Tothe solution (B), an aqueous solution about 4.6 to give the solution(B). An aqueous solution of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (10 mg/ml;1.27 ml) was added to the solution (B) and adjusted with 0.1Nhydrochloric acid to a pH of 4.6, followed by stirring at a temperaturebelow 5° C. for about 15 hours. The resulting mixture was admitted in adialyzing tube and subjected to dialysis in a conventional manner for 24hours, followed by centrifugation and lyophilization to give the humanserum albumin-combined BMTS as white cotton-like crystals. The crystals(67 mg) were dissolved in 0.2M acetate buffer (pH, 2.64; 5 ml) aspreviously eliminated dissolved oxygen therefrom, and 0.1 mM aqueoussolution of stannous chloride (2.0 ml) and ascorbic acid (1.18 mg) wereadded thereto. The resultant solution was passed through a filter havinga pore size of 0.22 μm, and 1.5 ml of the filtrate were filled in avial, of which the inside was flushed with nitrogen, to obtain anon-radioactive carrier as a slightly pale yellow, transparent solution.The above operations were effected under sterile conditions.

EXAMPLE 4 Preparation of human serum albumin-combined BETS as anon-radioactive carrier

BETS (4 mg) was dissolved in dimethylformamide (2 ml), an equimolaramount of glutaraldehyde (25% solution) to BETS was added thereto, andthe resultant mixture was stirred at room temperature for 15 minutes tomake the solution (A). Separately, human serum albumin (lyophilized; of1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (50 mg/ml; 1.5 ml)was added, and the resultant mixture was adjusted with 0.1N hydrochloricacid to a pH of about 4.6, followed by stirring at a temperature below5° C. for about 3 hours. The reaction mixture was chromatographed onSephadex G-50 and eluted with 0.01M phosphate buffer-0.15M sodiumchloride solution (pH, 7.4). The eluate was diluted with 0.01Mphosphate-0.15M sodium chloride solution to make a concentration of 5.0mg/ml of urokinase. The dilution was passed through a filter having apore size of 0.22 μm, and 1.5 ml of the filtrate were filled in a vialto obtain a non-radioactive carrier as a slightly pale yellowtransparent solution. The above operations were effected under sterileconditions.

EXAMPLE 6 Preparation of ^(99m) Tc-labeled, human serum albumin-combinedBMTS as a radioactive diagnostic agent

The human serum albumin-combined BMTS (solution) obtained in Example 3(1.5 ml) was admixed with a physiological saline solution (0.5 ml)containing ^(99m) Tc (15 mCi) in the form of pertechnetate, followed bystirring to give an aqueous solution containing the ^(99m) Tc-labeled,human serum albumin-combined BMTS useful as a radioactive diagnosticagent. This solution was pale yellow, transparent and had around pH 3.2.

EXAMPLE 7 Preparation of ⁶⁷ Ga-labeled, human serum albumin-combinedBETS as a radioactive diagnostic agent

The human serum albumin-combined BETS (lyophilized powder) obtained inExample 4 was dissolved in 0.2M acetate buffer (pH, 4.0; 1.5 ml), and0.01N hydrochloric acid (0.5 ml) containing ⁶⁷ Ga (2 mCi) in the form ofgallium chloride was added thereto to give an aqueous solutioncontaining the ⁶⁷ Ga-labeled, human serum albumin-combined BETS usefulas a radioactive diagnostic agent. This solution was slightly paleyellow, transparent and had around pH 3.7.

EXAMPLE 8 Preparation of ¹¹¹ In-labeled, urokinase-combined BMTS as aradioactive diagnostic agent

The urokinase-combined BMTS (solution) obtained in Example 5 (1.5 ml)was admixed with 0.1N hydrochloric acid (0.5 ml) containing ¹¹¹ In (0.5mCi) in the form of indium chloride to give an aqueous solutioncontaining the ¹¹¹ In-labeled, urokinase-combined BMTS useful as aradioactive diagnostic agent. This solution was slightly pale yellow,transparent and had around pH 6.0.

EXAMPLE 9 Properties of ^(99m) Tc-labeled, human serum albumin-combinedBMTS

In order to examine the labeling efficiency of the ^(99m) Tc-labeled,human serum albumin-combined BMTS obtained in Example 6, its aqueoussolution was subjected to thin layer chromatography using silica gel asa retention material and methylethylketone or 85% methanol as adeveloping solvent, and scanning was carried out by the use of aradio-chromato-scanner. Irrespective of the kind of the developingsolvent, the radioactivity was recorded as a single peak at the originalpoint. Any peak due to a radioactive impurity such as free pertechnetateion (Rf=1.0 in case of using methylethylketone; Rf=0.8-0.9 in case ofusing 85% methanol) was not recognized.

Then, the ^(99m) Tc-labeled, human serum albumin-combined BMTS obtainedin Example 6 was subjected to electrophoresis (1.7 mA/cm; 15 minutes)using a Veronal-Veronal Na solution (pH 8.6) as a developing solvent anda cellulose acetate membrane as an electrophoretic membrane, andscanning was effected by the use of a radiochromato-scanner. Theradioactivity was recognized as a single peak at the locus 1.8 cmdistant from the original line to the positive side. This locus was thesame as that of the coloring band of human serum albumin with Ponceau3R.

From the above results, it may be said that the ^(99m) Tc-labeled, humanserum albumin-combined BMTS obtained in Example 6 has a labelingefficiency of nearly 100%, and its electric charge is substantially thesame as that of human serum albumin.

EXAMPLE 10 Behaviors of ^(99m) Tc-labeled, human serum albumin-combinedBMTS in rabbits

The ^(99m) Tc-labeled, human serum albumin-combined BMTS obtained inExample 6 (0.3 ml) was administered intravenously to each of rabbitshaving a bypass formed by a cannula at the carotid artery through theear vein, and the variation of the blood level with the lapse of timewas examined by measurement of the radioactivity at the bypass.

The results are shown in Table 1 wherein the blood level at eachmeasuring time (with correction for physical attenuation of ^(99m) Tc)is indicated by a relative value (in average) to that immediately afterthe administration which is taken as 1.0.

                  TABLE 1                                                         ______________________________________                                        Variation of blood level in rabbits                                           Time after                                                                    administration                                                                (hours)   0     0.25     0.5  1      2    3                                   ______________________________________                                        Relative value                                                                          1.0   0.96     0.90 0.80   0.68 0.62                                ______________________________________                                    

EXAMPLE 11 Behaviors of ^(99m) Tc-labeled, human serum albumin-combinedBMTS in rats

The ^(99m) Tc-labeled, human serum albumin-combined BMTS obtained inExample 6 (0.1 ml) was administered intravenously to each of female ratsof SD strain at the tail vein, and the variation of the blood level withthe lapse of time was recorded. For the control, the same examination asabove was carried out by the use of conventional ^(99m) Tc-labeled,human serum albumin and conventional ¹³¹ I-labeled, human serum albumin.

The results are shown in Table 2 wherein the blood level at eachmeasuring time is indicated by an absolute value (in average).

                  TABLE 2                                                         ______________________________________                                        Variation of blood level in rats (%/g)                                                   Time after administration (hours)                                  Agent tested 0.25   0.5      1     2    3                                     ______________________________________                                        .sup.99m Tc-labeled,                                                                       7.79   6.97     6.33  5.47 5.07                                  human serum                                                                   albumin-combined                                                              BMTS                                                                          Conventional 5.49   3.95     3.68  --   2.51                                  .sup.99m Tc-labeled,                                                          human serum                                                                   albumin (Commer-                                                              cial product A)                                                               Conventional 3.78   3.15     2.16  --   1.88                                  .sup.99m Tc-labeled,                                                          human serum                                                                   albumin (Commer-                                                              cial product B)                                                               Conventional 7.01   5.93     5.64  --   4.59                                  .sup.131 I-labeled,                                                           human serum                                                                   albumin                                                                       ______________________________________                                    

The distribution of the ^(99m) Tc-labeled, human serum albumin-combinedBMTS obtained in Example 6 in the organs of rats with the lapse of timewas also observed, and the results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Distribution of .sup.99m Tc-labeled, human serum                              albumin-combined BMTS in organs of rats                                       with lapse of time (average in 5 animals; %/g)                                        Time after administration (hours)                                     Organs    0.25      0.5    1       2    3                                     ______________________________________                                        Blood     7.79      6.97   6.33    5.47 5.07                                  Liver     1.39      1.43   1.38    1.20 1.43                                  Spleen    0.83      0.88   0.90    0.64 0.84                                  Lung      1.49      1.40   1.25    0.95 1.16                                  Kidneys   1.64      2.00   2.16    2.44 2.69                                  Heart     1.69      1.35   1.60    1.00 0.97                                  Stomach   0.40      0.70   0.89    0.74 0.92                                  Small intestine                                                                         0.40      0.56   0.62    0.75 0.97                                  Large intestine                                                                         0.18      0.15   0.18    0.17 0.23                                  ______________________________________                                         Note:                                                                         Body weight standardized to 190 grams. Organs contained blood.           

From the results in Examples 10 and 11, it is understood that the ^(99m)Tc-labeled, human serum albumin-combined BMTS can maintain a remarkablyhigh blood level for a long period of time in comparison withconventional ^(99m) Tc-labeled, human serum albumin and conventional ¹³¹I-labeled, human serum albumin. It is also understood that the ^(99m)Tc-labeled, human serum albumin-combined BMTS is quite stable in aliving body and gives a relatively low radioactivity at various organsin comparison with the blood level. Thus, the ^(99m) Tc-labeled, humanserum albumin-combined BMTS is quite suitable for the use in nuclearmedical diagnosis aiming at recording, dynamic study and quantitativemeasurement of the blood circulation system.

EXAMPLE 12 Properties of ⁶⁷ Ga-labeled, human serum albumin-combinedBETS

In order to examine the labeling efficiency of the ⁶⁷ Ga-labeled, humanserum albumin-combined BETS obtained in Example 7, it was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using a Veronal-Veronal Nasolution (pH 8.6) as a developing solvent and a cellulose acetatemembrane as an electrophoretic membrane, and scanning was effected bythe use of a radiochromato-scanner. The radioactivity was recognized asa single peak at the locus 1.8 cm distant from the original line to thepositive side. This locus was the same as that of the coloring band ofhuman seum albumin with Ponceau 3R.

From the above results, it may be said that the ⁶⁷ Ga-labeled, humanserum albumin-combined BETS obtained in Example 7 has a labelingefficiency of nearly 100%, and its electric charge is substantially thesame as that of human serum albumin.

EXAMPLE 13 Properties of ¹¹¹ In-labeled, urokinase-combined BMTS

The enzymatic activity of the ¹¹¹ In-labeled, urokinase-combined BMTSwas measured by the ester decomposition process usingN-α-acetyl-L-lysine methyl ester to be 98% based on purified urokinaseas the starting material.

From the above results, it may be said that the ¹¹¹ In-labeled,urokinase-combined BMTS retains substantially the enzymatic activity ofthe starting purified urokinase and shows no material difference fromurokinase itself in the behavior in a living body.

EXAMPLE 14 Stability of human serum albumin-combined BMTS

The human serum albumin-combined BMTS (solution) obtained in Example 3was stored in a refrigerator at 4 to 8° C. for 30 days and then treatedwith ^(99m) Tc according to the procedure as in Example 6 to give anaqueous solution containing the ^(99m) Tc-labeled, human serumalbumin-combined BMTS. With this solution, thin layer chromatography andelectrophoresis were carried out according to the procedure as inExample 9 and also behaviors in rats were examined according to theprocedure as in Example 11. The results were substantially the same asin Examples 9 and 11. Thus, it may be said that no material change isproduced in the human serum albumin-combined BMTS by the storage for 30days.

EXAMPLE 15 Stability of human serum albumin-combined BETS

The human serum albumin-combined BETS (lyophilized powder) obtained inExample 4 was stored in a refrigerator at 4° to 8° C. for 30 days andthen treated with ⁶⁷ Ga according to the procedure as in Example 7 togive an aqueous solution containing the ⁶⁷ Ga-labeled, human serumalbumin-combined BETS. With this solution, the electrophoresis wascarried out according to the procedure as in Example 12. Theradioactivity was recognized as a single peak, and its locus wasconfirmed to be the same as that of human serum albumin by coloring withPonceau 3R. Thus, it may be said that no material change is produced inthe human serum albumin-combined BETS by the storage for 30 days.

EXAMPLE 16 Stability of ^(99m) Tc-labeled, human serum albumin-combinedBMTS

An aqueous solution containing the ^(99m) Tc-labeled, human serumalbumin-combined BMTS obtained in Example 6 was stored at roomtemperature (24°-27° C.) for 36 hours. With this solution, thin layerchromatography and electrophoresis were carried out according to theprocedure as in Example 9 and also behaviors in rats were examinedaccording to the procedure as in Example 11. The results weresubstantially the same as in Examples 9 and 11. Thus, it may be saidthat no material change is produced in the ^(99m) Tc-labeled, humanserum albumin-combined BMTS by the storage of 36 hours.

EXAMPLE 17 Stability of ⁶⁷ Ga-labeled, human serum albumin-combined BETS

An aqueous solution containing the ⁶⁷ Ga-labeled, human serumalbumin-combined BETS obtained in Example 7 was stored at roomtemperature (24°-27° C.) for 72 hours. With this solution,electrophoresis was carried out according to the procedure as in Example12. The radioactivity was recognized as a single peak, and its locus wasconfirmed to be substantially the same as that of human serum albumin bycoloring with Ponceau 3R. Thus, it may be said that no material changeis produced in the ⁶⁷ Ga-labeled, human serum albumin-combined BETS bythe storage of 72 hours.

EXAMPLE 18 Toxicity of non-radioactive carriers

The non-radioactive carriers obtained in Examples 3 to 5 (perfectlydissolved in 0.2M acetate buffer in case of the non-radioactive carrierobtained in Example 4) were administered intravenously to groups of maleand female rats of SD strain, each group consisting of 10 animals, at adose of 1 ml per 100 grams of the body weight (corresponding to 400times the expected dose to human beings) and also to groups of male andfemale mice of ICR strain, each group consisting of 10 animals, at adose of 0.5 ml per 10 grams of the body weight (corresponding to 2000times the expected dose to human beings). As the control, the samevolume of a physiological saline solution as above was intravenouslyadministered to the separate groups of the same animals as above.

The animals were fertilized for 10 days, and the variation in bodyweight during that period was recorded. No significant difference wasrecognized between the medicated groups and the control groups.

After 10 days from the administration, all the animals were sacrificedand subjected to observation of the abnormality in various organs. But,no abnormality was seen in any of the animals.

From the above results, it may be said that the toxicity of thenon-radioactive carriers of the invention is extremely low.

EXAMPLE 19 Toxicity of the radioactive diagnostic agent

The ^(99m) Tc-labeled, human serum albumin-combined BMTS obtained inExample 6 was subjected to attenuation of the radioactivity to anappropriate extent, and the resultant product was subjected to test fortoxicity in the same manner as in Example 18. No significant differencewas recognized between the medicated groups and the control groups. Inall the animals sacrificed after 10 days from the administration, noabnormality was observed in their organs. Thus, it may be said that theradioactive diagnostic agent of the invention does not produce anymaterial toxicity in tested animals even when administered in such alarge dose as corresponding to 300 to 1500 times the expected dose tohuman beings.

What is claimed is:
 1. A compound of the formula: ##STR3## wherein R¹and R² are each hydrogen, C₁ -C₃ alkyl or phenyl.
 2. The compoundaccording to claim 1, wherein R¹ and R² are each methyl.
 3. The compoundaccording to claim 1, wherein R¹ and R² are each ethyl.